The invention relates to the field of compositions, methods, and apparatus for synthesizing and characterizing polymers, including polynucleotides.
Synthesis of macromolecules for use in biological studies, in the preparation of drugs for treatment of disease and disorders, for example polynucleotides encoding antibodies, growth factors, cytokines, or the like, for use in diagnoses of conditions, disease and disorders, currently utilize multi-molecular systems, such as chemical, biochemical, and cellular systems, that frequently require purified enzymes, reagents, and co-factors, thereby incurring significant cost in both monetary and temporal measures. In general, once used, most of the reagents are rendered unusable, being contaminated and/or degraded during the synthesis processes.
There is therefore a need in the art to provide a simple system wherein single monomers may be combined to create polymers under low energy constraints using few and/or simple, inexpensive, reagents.
The following studies on the origins of enzymatic and non-enzymatic hydrolysis of covalent and ionic bonds in a self-organizing system (i.e. “life”), usually under standard temperature and pressure (STP) or at temperature and pressure extremes, such as might occur in a prebiotic environment or in geological formations, disclose methods that have been shown to be particularly relevant to identifying how this need may be fulfilled.
Several studies have investigated possible pathways for the synthesis and degradation of RNA under hydrothermal conditions (White (1984) Nature 310: 430-432; Larralde et al. (1995) Proc. Natl. Acad. Sci. USA 92: 8158-8160; Miller and Lazcano (1995) J. Mol. Evol. 41: 689-692; Kawamura et al. (1997) Viva Origino 25: 177-190; Levy and Miller (1998) Proc. Natl. Acad. Sci. USA 95: 7933-7938; Kawamura (2004) Intl. J. Astrobiol. 3: 301-309). Kawamura's results suggest that it is possible for synthesis of phosphodiester bonds to occur in hot aqueous solutions if chemically activated monomers and catalysts are present. We have previously shown that lipid vesicles can encapsulate oligomerization reactions (Chakrabarti et al. (1994) J. Mol. Evol. 39: 555-559) and can also provide an organizing template for the non-enzymatic polymerization of thioglutamic acid to peptides (Zepik et al. (2007) Orig. Life Evol. Biosph. Mar. 25, 2007 (E-publication ahead of print)).
Because polymerization by condensation is thermodynamically unfavorable in aqueous solutions, an energy source is required to drive phosphodiester bond formation. Imidazole esters of mononucleotides are commonly used as activated monomers and readily assemble on RNA templates to produce complementary RNA strands up to 30 nucleotides in length (Inoue and Orgel (1983) Science 219: 859-862; Orgel (1998) Orig. Life Evol. Biosphere 28: 227-234). Huang and Ferris (Huang and Ferris (2003) Chem. Commun. 21: 1458-1461) and Ferris (2002, supra) found that the mineral surfaces of montmorillonite clay can organize chemically-activated mononucleotides so that RNA-like polymer chains in the 6-14 mer range are synthesized in the absence of templates, and up to 40-50 mers if a 10 mer is added as a primer or 1-methyladenine is used to activate the phosphate group of mononucleotides (Huang and Ferris (2006) J. Am. Chem. Soc. 128: 8914-8919).
These conditions are useful models for investigating non-enzymatic polymerization mechanisms, but a plausible source of activated monomers in the prebiotic environment remains elusive. For this reason we are investigating other conditions that could drive polymer synthesis. We first note that phosphodiester bond formation is a relatively low-energy reaction. It was estimated that the standard free energy of synthesis is +5.3 kcal/mol (Dickson et al. (2000) J. Biol. Chem. 275: 15828-15831) that is similar to that of glucose-1-phosphate formation (+5.0 kcal/mol) from glucose and phosphate in solution. Thus, it should be possible to drive phosphodiester bond formation in the absence of activated substrates by producing conditions in which water can be removed from the reactants. More recently, Kawamura (2002, Anal. Sci. 18: 715-716) developed a method to monitor RNA synthesis and degradation of RNA under simulated hydrothermal vent conditions, and demonstrated that the rate of phosphodiester bond formation was faster than the rate of decomposition at 100° C., but at higher temperature ranges (200 and 300° C.) degradation rates far exceeded synthesis. These results set an upper limit on thermal conditions for the origin of life, but also made it clear that there are no thermodynamic or kinetic barriers to RNA synthesis and stability in hyperthermophilic organisms like the chemolithoautotrophic archaeon Pyrolobus fumarii, which has been shown to be able to grow at 110° C. (Stetter (1999) FEBS Lett. 452: 22-25). Other extremophiles have also been found not only to survive but to thrive at such high temperatures (Stetter (1982) Nature 300: 258-260; Kashefi and Lovely (2003) Science 301: 934).
There is currently a need to provide compositions and methods that can be used in synthesis of polymers, including polynucleotides and polypeptides.